Crown ether derivatives

• US 7,129,346 B2
• Filed: 08/04/2003
• Issued: 10/31/2006
• Est. Priority Date: 12/20/2000
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Gee, Kyle R., Martin, Vladimir V.
• Primary Examiner(s): Kifle, Bruck
• Application Number:
  US10/634,336
• Publication Number:
  US 20040096978A1
• Time in Patent Office: 1,184 Days
• Field of Search: 540/465, 540/468
• US Class Current: 540/465
• CPC Class Codes: C07D 273/00 Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 57/10 Metal complexes of organic compounds not being dyes in uncomplexed form
• CPC Class Codes: C09B 69/00 Dyes not provided for by a single group of this subclass
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH

Abstract

The invention describes crown ether chelators, including crown ethers having the formula

and aza-substituted and thia-substituted analogs thereof. These crown ethers may be substituted by a dye moiety, a chemically reactive group, a conjugated substance, or a combination thereof. Chelators that are substituted by fluorescent dyes are particularly useful as indicators for metal cations, particularly Na⁺ and K⁺ ions, and particularly where binding of the target ion results in a change in the fluorescence properties of the indicator that can be correlated with the ion concentration. Methods are provided for utilizing reactive groups on the chelators for conjugation to dyes, lipids and polymers and methods for enhancing entry of the indicators into living cells.

Claims

1. A crown ether chelating compound having formula:

2. The compound according to claim 1, wherein at least one of said R¹, R⁷, R⁸, R⁹, R¹⁰, R¹⁹ or R²⁰ is -L-Rx, -L-Sc or -L-DYE or R⁸ in combination with R⁹ form a fused DYE.

3. The compound according to claim 1, wherein said L is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1–

20 nonhydrogen atoms selected from the group consisting of C, N, P, O and S; and

are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

4. The compound according to claim 1, wherein said—

Rx is selected from the group consisting of carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine and a maleimide.

5. The compound according to claim 1, wherein said—

Sc is selected from the group consisting of an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.

6. The compound according to claim 1, wherein said—

DYE is selected from the group consisting of xanthene, borapolyazaindacene, carbocyanine, benzofuran, quinazolinone, indole, a benzazole, oxazine, and coumarin.

7. The compound according to claim 6, wherein said—

DYE moiety is independently substituted by a lipophilic group.

8. The compound according to claim 7, wherein said lipophilic group is an AM or acetate ester.

9. The compound according to claim 1, wherein said compound is selected from the group consisting of

10. The compound according to claim 9, wherein said DYE is selected from the group consisting of borapolyazaindacene, xanthene and indole.

11. The compound according to claim 9, wherein said DYE moiety is independently substituted by a lipophilic group.

12. The compound according to claim 11, wherein said lipophilic group is an AM or acetate ester.

13. The compound according to claim 9, wherein R⁷, R⁸, R⁹, R¹⁰, R¹⁹ and R²⁰, when present, are H.

14. The compound according to claim 13, wherein R¹ is C₁–

C₆ alkyl that is substituted one or more times by amino (—

NR¹⁷R¹⁸), —

(C═

O)—

O—

R¹⁶ or —

(C═

O)—

NR¹⁷R¹⁸.

15. The compound according to claim 14, wherein said R¹ is methyl or ethyl.

16. The compound according to claim 15 wherein said R¹⁶ is selected from the group consisting of H, C₁–

C₆ alkyl, benzyl, a biologically compatible esterifying group, and a biologically compatible salt.

17. The compound according to claim 16 wherein said R¹⁶ is methyl.

18. The compound according to claim 14 wherein said R¹⁷ and R¹⁸ are each methyl.

19. A compound having formula:

20. The compound according to claim 19, wherein said R⁷, R⁸, and R¹⁰ are H.

21. The compound according to claim 20, wherein said R¹⁹ and R²⁰ are H.

22. The compound according to claim 21, wherein said R¹⁶ is methyl or a biologically compatible esterifying group.

23. The compound according to claim 19 wherein said R¹⁶, R¹⁹ or R²⁰ is —

L-Sc.

24. A composition comprising:

a) a compound having the formula

25. The composition according to claim 24, wherein said metal ion is selected from the group consisting of Na⁺, Li⁺, K⁺, Ca⁺, Zn⁺ and Rb⁺.

26. A method for binding a target metal ion in a sample, comprising steps of:

a) contacting said sample with a metal chelating compound having the formula

27. The method according to claim 26, wherein said method further comprises illuminating said metal chelating compound with a suitable light source whereby said target ion is detected with the proviso that at least one of R¹, R⁴, R⁷, R⁸, R⁹, R¹⁰, R¹⁹ or R²⁰ is —

L-DYE or at least two of R⁷–

R¹⁰ or R¹⁹ and R²⁰, taken in combination, form a fused DYE.

28. The method according to claim 27, wherein said target metal ion is selected from the group consisting of Na⁺, Li⁺, K⁺, Ca⁺, Zn⁺ and Rb⁺.

29. The method according to claim 28, wherein said target metal ion is Na⁺.

30. The method according to claim 28, wherein said sample comprises living cells, cellular components, proteins, peptides, buffer solutions or biological fluids.

31. A method for binding and detecting target ions in a live cell, said method comprises:

a) contacting a sample of live cells with a crown ether compound with the proviso that said compound comprise a DYE moiety and at least one lipohilic group, wherein the crown ether compounds has the formula

32. The method according to claim 31, wherein said DYE moiety is substituted by a lipophilic group.

33. The method according to claim 32 wherein said lipophilic group is an AM or acetate ester.